Staphylococcus aureus antigen-containing whole cell vaccine

ABSTRACT

A negatively-charged  S. aureus  antigen contains β-hexosamine as a major carbohydrate component.  S. aureus  strains that carry the antigen account for nearly all of the clinically significant strains of  S. aureus  that are not Type 5 or Type 8 strains. The antigen can be used in combination with  S. aureus  Type 5 polysaccharide antigen and  S. aureus  Type 8 polysaccharide antigen to provide nearly 100% coverage of  S. aureus  infection. The antigen and antibodies to the antigen are useful in kits and assays for diagnosing  S. aureus  infection. A whole cell vaccine of cells that contain the antigen is particularly useful in the treatment of mastitis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuing application, under 35 U.S.C. §120, ofapplication Ser. No. 09/102,214, filed Jun. 22, 1998, now U.S. Pat. No.6,194,161, which is a continuation-in-part of and claims priority toapplication Ser. No. 08/712,438, filed Sep. 11, 1996, now U.S. Pat. No.5,770,208.

BACKGROUND OF THE INVENTION

The present invention relates to a novel Staphylococcus aureus antigen,and to a method for obtaining and using the antigen.

S. aureus causes several diseases in animals and in humans by variouspathogenic mechanisms. The most frequent and serious of these diseasesare bacteremia and its complications in hospitalized patients. Inparticular, S. aureus can cause wound infections and infectionsassociated with catheters and prosthetic devices. Serious infectionsassociated with S. aureus bacteremia include osteomyelitis, invasiveendocarditis and septicemia. The problem is compounded by multipleantibiotic resistance in hospital strains, which severely limits thechoice of therapy. In addition, S. aureus is a major cause of mastitisin dairy and beef cattle, where the infection causes a major loss ofincome.

A S. aureus vaccine would provide a solution for the problem ofantibiotic resistance. At least eight different serotypes of S. aureushave been identified using polyclonal and monoclonal antibodies tocapsular polysaccharide (CPS). Karakawa et al., J. Clin. Microbiol.22:445 (1985). The contents of this document and all others listedherein are incorporated herein by reference.

Surveys have shown that approximately 85-90% of human clinical isolates,and a comparable, although somewhat lower percentage of animal clinicalisolates, are capsular polysaccharide Type 5 or Type 8. An individualvaccinated with a vaccine containing Type 5 and Type 8 CPS antigenswould be protected from infection by 85-90% of clinically-significant S.aureus strains, but a significant risk of infection still would exist. Avaccine containing antigens from the other six serotypes theoreticallycould provide 100% protection, but would require production andpurification of six additional components. This would be untenable froma practical standpoint. On the other hand, an antigen common to theisolates not typeable as Type 5 or Type 8 would enable production of avaccine containing only three antigens.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a wholecell vaccine of cells that carry an antigen that is common to a largenumber of clinically-significant S. aureus strains, particularly onethat is common to strains associated with infections in animals,particularly cattle.

It is a further object of the invention to provide a whole cell vaccinefor prevention or treatment of infections in animals, particularlymastitis in cattle.

It is another object of the invention to provide a hyperimmune globulincomposition that contains antibodies directed against bacteria thatcarry an antigen that is common to a large number ofclinically-significant S. aureus strains, particularly one that iscommon to strains associated with infections in animals, particularlycattle.

It is yet another object of the invention to provide a hyperimmuneglobulin composition that is effective in treatment of infections inanimals, particularly mastitis.

In accordance with these and other objects according to the invention,there is provided a whole cell vaccine comprising cells from a strain ofStaphylococcus aureus that carries an antigen that comprises β-linkedhexosamine, that contains no O-acetyl groups detectable by nuclearmagnetic resonance spectroscopy and that reacts with antibodies to ATCC55804. Also provided is a composition comprising the whole cell vaccine,and a sterile, pharmaceutically-acceptable carrier therefor. The vaccinecan be administered to a human or animal subject to provide protectionagainst S. aureus infection. It is particularly useful in preventingmastitis in animals.

An immunotherapeutic agent against S. aureus infection, particularlyagainst mastitis in animals, can be prepared by immunizing human oranimal subjects with a composition according to the invention,collecting plasma from the immunized subjects, and harvesting a human orveterinary hyperimmune globulin that contains antibodies directedagainst S. aureus from the collected plasma. The hyperimmune globulincontains antibodies directed against the β-linked hexosamine antigen. Animmunotherapy method comprises a step of administering this hyperimmuneglobulin to a human or animal subject, especially an animal withmastitis, to prevent or treat infection.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1 b and 1 c show the NMR spectra for each of 336, Type 8 andType 5 S. aureus antigens, respectively.

FIG. 2 is a bar graph showing the ability of 336 conjugate IgG tomediate opsonophagocytosis of a representative strain of S. aureus thatcarries the 336 antigen.

FIG. 3 is a bar graph showing the ability of 336 whole cell IgG tomediate opsonophagocytosis of a representative strain of S. aureus thatcarries the 336 antigen.

FIG. 4 is a bar graph showing the effect of absorption with T5 CPSantigen or 336 antigen on the ability of 336 whole cell IgG to mediateopsonophagocytosis of a representative strain of S. aureus that carriesthe 336 antigen.

DESCRIPTION OF PREFERRED EMBODIMENTS

It has been discovered that virtually all strains of S. aureus that arenot typeable as Type 5 or Type 8 have in common an antigen, here denoted“the 336 antigen.” When combined with Type 5 and Type 8 antigens, the336 antigen represents the basis for a vaccine that provides almostcomplete protection against infection by clinically significant S.aureus isolates. In this regard, a “clinically significant” isolate isan isolate that is pathogenic, in either humans or animals.

More particularly, typing of isolates obtained from various sources hasshown that approximately 60% of human isolates are Type 8, approximately30% are Type 5 and that nearly all of the remaining 10% of isolates areType 336, as shown in Table 1. Less than 1% of the isolates are nottypeable as one of these three types.

TABLE 1 Typing of human clinical isolates Source Total Type 5 Type 8Type 336* Not typeable Canadian 350 109 206 34 1 (31.5%) (58.8%) (9.7%)(0.3%) Cystic 147 29 92 19 — Fibrosis (19.7%) (62.6%) (12.2%) *Arepresentative strain of S. aureus that carries the 336 antigen has beendeposited under the Budapest Treaty with the American Type CultureCollection, and has been given Accession No. 55804.

Notably, the present inventors obtained 27 human clinical isolates thatwere not typeable as either Type 5 or Type 8 strains of S. aureus, andthat were characterized as being methicillin-resistant strains. All ofthe 27 strains reacted very strongly with 336 antigen conjugate antibodysera, and thus were typeable as strains that contain 336 antigen.

Typing of bovine mastitis isolates obtained from various sources hasshown that approximately 23% of human isolates are Type 8, approximately22% are Type 5 and that 54% of isolates are Type 336, as shown in Table2. Thus, 97.5% of isolates that previously were not typeable aretypeable as Type 336. Less than 2% of the isolates are not typeable asone of these three types. Therefore, a trivalent whole cell vaccine ofType 5, Type 8 and 336 is particularly indicated for treatment andprevention of veterinary S. aureus infections.

TABLE 2 Typing of bovine mastitis isolates Not Source Total Type 5 Type8 Type 336* typeable Europe 102 35 33 29 5 (34.3%) (32.4%) (28.4%)(4.9%) United States 336 59 68 207 2 (17.6%) (20.2%) (61.6%) (0.6%)Total 438 94 101 236 7 (21.5%) (23%)  (53.9%) (1.6%)

Antibodies to the 336 antigen do not cross-react with polysaccharidesisolated from any of S. aureus Type 5, Type 8, Type 4, K73 (a Type 5variant strain) or S. epidermidis. The 336 antigen therefore istype-specific, that is, it produces a single band with only thehomologous-type antiserum.

The 336 antigen can be obtained in recoverable amount, from certain S.aureus isolates cultured pursuant to the protocols described herein, insubstantially pure form. In particular, purified antigen contains lessthan 1% protein and less than 1% nucleic acids. A “recoverable” amountin this regard means that the isolated amount of the antigen isdetectable by a methodology less sensitive than radiolabeling, such asimmunoassay, and can be subjected to further manipulations involvingtransfer of the antigen per se into solution.

To obtain the 336 antigen, a 336 isolate according to the inventionfirst is grown on a Columbia Broth agar plate supplemented withMgCl₂/CaCl₂ and then transferred to a 2% NaCl/Columbia starter flask. A50-liter fermentor that contains the same medium is inoculated with thestarter flask. Cells are fermented for 16-24 hours. Followingfermentation, cells are killed with 2% final concentration of phenol toethanol (1:1) and then centrifuged to separate the cells from thesupernatant. Antigen is extracted from cell paste. Some 336 antigen ispresent in the supernatant, but the amount is insignificant as comparedto the amount found in the cell paste. Because of the low yield, and therisk of hexose contamination from the media, extraction from supernatantis not preferred.

Enzyme treatments of cell paste with lysostaphin, DNase, RNase andprotease, followed by sequential precipitation with 25-75% coldethanol/CaCl₂, results in a crude antigen extraction. The crude materialis redissolved in water, dialyzed and lyophilized. The lyophilizedmaterial is dissolved in buffer and loaded onto a separatory columnequilibrated with the same buffer. The column is washed with 0.15 M NaClloading buffer and then eluted with a 0.15-0.4 M NaCl gradient. Most ofthe antigen according to the invention elutes at 0.32 to 0.35 M NaCl.Fractions containing antigen are pooled, dialyzed, concentrated, andlyophilized. The separation can be repeated to obtain betterpurification. The crude antigen is treated with lysozyme and purified bysize on a suitable column and the 336 positive fractions are thenpooled, concentrated, dialyzed and lyophilized.

Analysis of purified 336 antigen by gas liquid chromatography (GLC)shows the presence of glucosamine as a major glycosyl component. This isconfirmed by sugar analysis on a Dionex system. 1H-NMR spectroscopy ofthe 336 antigen indicates the presence of β-linked hexosamine as a majorcarbohydrate component. The antigen additionally comprises a componentthat is responsible for an observed negative charge displayed by the 336antigen.

A comparison of the NMR spectra for each of the 336, Type 5 and Type 8S. aureus antigens, as shown in FIGS. 1a, 1 b and 1 c, confirms that the336 antigen is chemically distinct from both the Type 5 and Type 8 S.aureus antigens. The structures of Types 5 and 8 polysaccharide antigenshave been elucidated by Moreau et al., Carbohydr. Res. 201:285 (1990);and Fournier et al., Infect. Imm. 45:87 (1984). Both have FucNAcp intheir repeat unit as well as ManNAcA which can be used to introduce asulfhydryl group. The structures are as follows:

By contrast, the main carbohydrate component of the 336 antigen isβ-linked hexosamine.

Induction of bacteremia in laboratory animals requires extremely highnumbers of organisms or some previous maneuver to lower the hostresistance. In vitro phagocytosis, however, can be studied as acorrelate of protective immunity in vivo. In this model, the ability of336 antigen-specific monoclonal and polyclonal antibodies to opsonize S.aureus in vitro is measured by phagocytosis, according to the methoddescribed in Kojima et al., Infect. Dis. Immun. 58:2367-2374 (1990).

Antibodies induced by a 336 antigen vaccine facilitate type-specificphagocytosis. The in vitro phagocytosis assays thus indicate thatantibodies to the 336 antigen are protective against infection by S.aureus strains that carry the 336 antigen. Vaccines based on Type 5 andType 8 antigens previously have been shown to be protective againstinfection by Type 5 and Type 8 strains of S. aureus, respectively.Fattom et al. Inf. and Imm. 58:2367-2374 (1990) and loc. cit.64:1659-1665 (1996). A vaccine based on a combination of Type 5, Type 8and 336 antigen can be used to protect against infection from themajority of clinical S. aureus strains.

Preferably, a composition of the 336 antigen or of whole cellscontaining the 336 antigen according to the present invention “consistsessentially of” the 336 antigen, or cells that contain the 336 antigen.In this context, the phrase “consists essentially of” means that thecomposition does not contain any material that interferes withelicitation of an immune response to the 336 antigen (and to otherantigens, if present) when the composition is administered to a subjectas a vaccine, or with the antigen-antibody coupling characteristic of adiagnostic assay when the antigen is used in diagnosis. In oneembodiment, the composition contains Type 336, Type 5 and Type 8 S.aureus antigens.

The antigens according to the invention are useful in the production ofdiagnostic assays for detecting the presence of S. aureus antigen and/oranti-S. aureus antibody in a sample. S. aureus 336 antigen, or antibodyspecific to the S. aureus antigen, alone or in combination with antigenor antibody to one or both of Type 5 and Type 8 S. aureus antigens, ismixed with a sample suspected of containing S. aureus antigen orantibody and monitored for antigen-antibody binding. The antigen orantibody is labelled with a radioactive or enzyme label. In a preferredembodiment, antigen or antibody is immobilized on a solid matrix suchthat the antigen or antibody are accessible to complementary antibody orantigen contacting a surface of the matrix. The sample then is broughtinto contact with the surface of the matrix, and the surface ismonitored for antigen-antibody binding.

For example, the antigen or antibody can be used in an enzyme-linkedimmunosorbent assay (ELISA), in which antigen or antibody are bound to asolid phase and an enzyme-antibody or enzyme-antigen conjugate is usedto detect and/or quantify antibody or antigen present in a sample.Alternatively, a western blot assay can be used in which solubilized andseparated antigen(s) is bound to nitrocellulose paper. The antibody thenis detected by an enzyme or label-conjugated anti-immunoglobulin (Ig),such as horseradish peroxidase-Ig conjugate by incubating the filterpaper in the presence of a precipitable or detectable substrate. Westernblot assays have the advantage of not requiring purity greater than 50%for the desired antigen(s). Descriptions of ELISA and western blottechniques are found in Chapters 10 and 11 of Ausubel, et al. (eds.),CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons (1988), theentire contents of which are hereby incorporated by reference.

For use in a vaccine, or in stimulating the synthesis of type 336specific antibodies in immunized subjects, it is preferable to conjugatethe purified 336 antigen to an immunocarrier, usually a polypeptide orprotein, to improve the interaction between T and B cells for theinduction of an immune response against the antigen. This isparticularly important for vaccines intended for use in patients withreduced resistance. An immunocarrier enhances immunogenicity both foractive immunization and for preparing high-titered antisera involunteers for passive immunization. Suitable immunocarriers accordingto the present invention include tetanus toxoid, diphtheria toxoid,Pseudomonas aeruginosa Exotoxin A or its derivatives,recombinantly-produced non-toxic mutant strains of exotoxin A, asdescribed, for example, in Fattom et al., Inf. and Imm. 61:1023-1032(1993), as well as other proteins commonly used as immunocarriers.

Preferably, the antigen or antigen conjugate is administered without anadjuvant in order to avoid adjuvant-induced toxicity. If an adjuvant isused, it is preferred to use one which promotes the protective IgGsubtype 2 antibodies. Typical adjuvants include complete Freund'sadjuvant (CFA) and incomplete Freund's adjuvant (IFA). Dextran sulfatehas been shown to be a potent stimulator of IgG₂ antibody againststaphylococcal cell surface antigens, and also is suitable as anadjuvant.

The present invention also relates to the use of the 336 antigen toproduce polyclonal antibodies or monoclonal antibodies (mouse or human)that bind to or neutralize S. aureus strains that carry the 336 antigen.Protocols for producing these antibodies are described in Ausubel, etal. (eds.), Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, (Cold Spring Harbor, N.Y.)., Chapter 11; in METHODS OFHYBRIDOMA FORMATION 257-271, Bartal & Hirshaut (eds.), Humana Press,Clifton, N.J. (1988); in Vitetta et al., Immunol. Rev. 62:159-83 (1982);and in Raso, Immunol. Rev. 62:93-117 (1982).

Inoculum for polyclonal antibody production typically is prepared bydispersing the antigen-immunocarrier in a physiologically-tolerablediluent such as saline, to form an aqueous composition. Animmunostimulatory amount of inoculum, with or without adjuvant, isadministered to a mammal and the inoculated mammal is then maintainedfor a time period sufficient for the antigen to induce protectinganti-336 antigen antibodies. Boosting doses of the antigen-immunocarriermay be used in individuals that are not already primed to respond to theantigen.

Antibodies can include antibody preparations from a variety of commonlyused animals, e.g., goats, primates, donkeys, swine, rabbits, horses,hens, guinea pigs, rats, and mice, and even human antibodies afterappropriate selection, fractionation and purification. Animal antiseramay also be raised by inoculating the animals with formalin-killed 336strains of S. aureus, by conventional methods, bleeding the animals andrecovering serum or plasma for further processing.

The antibodies induced in this fashion can be harvested and isolated tothe extent desired by well known techniques, such as by alcoholfractionation and column chromatography, or by immunoaffinitychromatography; that is, by binding antigen to a chromatographic columnpacking like Sephadex™, passing the antiserum through the column,thereby retaining specific antibodies and separating out otherimmunoglobulins (IgGs) and contaminants, and then recovering purifiedantibodies by elution with a chaotropic agent, optionally followed byfurther purification, for example, by passage through a column of boundblood group antigens or other non-pathogen species. This procedure maybe preferred when isolating the desired antibodies from the sera orplasma of humans that have developed an antibody titer against thepathogen in question, thus assuring the retention of antibodies that arecapable of binding to the antigen. They can then be used in preparationsfor passive immunization against 336 strains of S. aureus.

A monoclonal antibody composition contains, within detectable limits,only one species of antibody combining site capable of effectivelybinding to the 336 antigen. Suitable antibodies in monoclonal form canbe prepared using conventional hybridoma technology.

To form hybridomas from which a monoclonal antibody composition of thepresent invention is produced, a myeloma or other self-perpetuating cellline is fused with lymphocytes obtained from peripheral blood, lymphnodes or the spleen of a mammal hyperimmunized with the 336 antigen. Itis preferred that the myeloma cell line be from the same species as thelymphocytes. Splenocytes are typically fused with myeloma cells usingpolyethylene glycol 1500. Fused hybrids are selected by theirsensitivity to HAT. Hybridomas secreting the antibody molecules of thisinvention can be identified using an ELISA.

A Balb/C mouse spleen, human peripheral blood, lymph nodes orsplenocytes are the preferred materials for use in preparing murine orhuman hybridomas. Suitable mouse myelomas for use in the presentinvention include the hypoxanthine-aminopterin-thymidine-sensitive (HAT)cell lines, a preferred myeloma being P3X63-Ag8.653. The preferredfusion partner for human monoclonal antibody production is SHM-D33, aheteromyeloma available from ATCC, Rockville, Md. under the designationCRL 1668.

A monoclonal antibody composition of the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules then can be isolated furtherby well known techniques.

Media useful for the preparation of these compositions are both wellknown in the art and commercially available, and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's Minimal essential medium supplemented with 20% fetal calfserum. An exemplary inbred mouse strain is the Balb/c.

Other methods of preparing monoclonal antibody compositions are alsocontemplated, such as interspecies fusions, since it is primarily theantigen specificity of the antibodies that affects their utility in thepresent invention. Human lymphocytes obtained from infected individualscan be fused with a human myeloma cell line to produce hybridomas whichcan be screened for the production of antibodies that recognize the 336antigen. More preferable in this regard, however, is a process that doesnot entail the use of a biological sample from an infected humansubject. For example, a subject immunized with a vaccine as describedherein can serve as a source for antibodies suitably used in an antibodycomposition within the present invention.

In a particularly preferred embodiment, monoclonal antibodies areproduced to the 336 antigen using methods similar to those described fortype-specific antibodies to S. aureus Type 5 and Type 8. The purifiedmonoclonal antibodies are characterized by bacterial agglutinationassays using a collection of clinical isolates.

The monoclonal and polyclonal antibody compositions produced accordingto the present description can be used by passive immunization to inducean immune response for the prevention or treatment of infection bystrains of S. aureus that carry the 336 antigen. In this regard, theantibody preparation can be a polyclonal composition. Such a polyclonalcomposition includes antibodies that bind to the 336 antigen, andadditionally may include antibodies that bind to the antigens thatcharacterize Type 5 and Type 8 strains of S. aureus. The polyclonalantibody component can be a polyclonal antiserum, preferably affinitypurified, from an animal which has been challenged with the 336 antigen,and preferably also with Type 5 and Type 8 antigens. Alternatively, an“engineered oligoclonal” mixture may be used, which is a mixture ofmonoclonal antibodies to the 336 antigen, and monoclonal antibodies Type5 and/or Type 8 antigens.

In both types of mixtures, it can be advantageous to link antibodiestogether chemically to form a single polyspecific molecule capable ofbinding to the 336 antigen and one or both of Type 5 and Type 8antigens. One way of effecting such a linkage is to make bivalentF(ab′)₂ hybrid fragments by mixing two different F(ab′)₂ fragmentsproduced, e.g., by pepsin digestion of two different antibodies,reductive cleavage to form a mixture of Fab′ fragments, followed byoxidative reformation of the disulfide linkages to produce a mixture ofF(ab′)₂ fragments including hybrid fragments containing a Fab′ portionspecific to each of the original antigens. Methods of preparing suchhybrid antibody fragments are disclosed in Feteanu, LABELED ANTIBODIESIN BIOLOGY AND MEDICINE 321-23, McGraw-Hill Int'l Book Co. (1978);Nisonoff, et al., Arch Biochem. Biophys. 93:470 (1961); and Hammerling,et al., J. Exp. Med. 128:1461 (1968); and in U.S. Pat. No. 4,331,647.

Other methods are known in the art to make bivalent fragments that areentirely heterospecific, e.g., use of bifunctional linkers to joincleaved fragments. Recombinant molecules are known that incorporate thelight and heavy chains of an antibody, e.g., according to the method ofBoss et al., U.S. Pat. No. 4,816,397. Analogous methods of producingrecombinant or synthetic binding molecules having the characteristics ofantibodies are included in the present invention. More than twodifferent monospecific antibodies or antibody fragments can be linkedusing various linkers known in the art.

An antibody component produced in accordance with the present inventioncan include whole antibodies, antibody fragments, or subfragments.Antibodies can be whole immunoglobulin of any class, e.g., IgG, IgM,IgA, IgD, IgE, chimeric antibodies or hybrid antibodies with dual ormultiple antigen or epitope specificities, or fragments, e.g., F(ab′)₂,Fab′, Fab and the like, including hybrid fragments, and additionallyincludes any immunoglobulin or any natural, synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. In particular, Fab molecules can be expressedand assembled in a genetically transformed host like E. coli. A lambdavector system is available thus to express a population of Fab's with apotential diversity equal to or exceeding that of subject generating thepredecessor antibody. See Huse, W. D., et al., Science 246:1275-81(1989).

The 336 antigen according to the present invention can be the activeingredient in a composition, further comprising a pharmaceuticallyacceptable carrier for the active ingredient, which can be used as avaccine to induce a cellular immune response and/or production in vivoof antibodies which combat S. aureus infection. In this regard, apharmaceutically acceptable carrier is a material that can be used as avehicle for administering a medicament because the material is inert orotherwise medically acceptable, as well as compatible with the activeagent, in the context of vaccine administration. In addition to asuitable excipient, a pharmaceutically acceptable carrier can containconventional vaccine additives like diluents, adjuvants, antioxidants,preservatives and solubilizing agents.

In an alternative embodiment, cells that carry the 336 antigen are usedin a whole cell vaccine. In this regard, a “whole cell vaccine” includesvaccines made using killed whole bacteria, bacterial lysates orderivatives of whole bacteria. Cells that carry the 336 antigen can beidentified and selected for use in the whole cell vaccine by usingantibodies to a strain known to carry the 336 antigen, and morepreferably by using monoclonal antibodies to isolated antigen asdescribed herein. In this regard, a simple slide agglutinationexperiment in which antibodies to 336 antigen are mixed with cells canbe used.

Deposited strain ATCC 55804 is a representative strain of S. aureus thatcarries the 336 antigen, and it can be used to produce antibodies usefulin identifying other strains that carry the 336 antigen. It is not,however, necessary, to use the deposited strain in order to produceeither the whole cell vaccine or antibodies useful in treating orpreventing S. aureus infections or in identifying other cells that carrythe 336 antigen. ATCC 55804 merely provides one immunologic means ofidentifying such cells.

As described for purified antigen vaccine above, the whole cell vaccinealso comprises a pharmaceutically acceptable carrier. The whole cellvaccine also optionally may contain conventional vaccine additives likediluents, adjuvants, antioxidants, preservatives and solubilizingagents. In a preferred embodiment, the whole cell vaccine contains cellsor derivatives of cells which carry the 336 antigen, in addition tocells or derivatives of cells which carry Type 5 and Type 8 antigens.

Vaccines according to the invention can be administered to a subject notalready infected with S. aureus, thereby to induce a S.aureus-protective immune response (humoral or cellular), particularly amastitis-protective response, in that subject. Alternatively, vaccineswithin the present invention can be administered to a subject in whichS. aureus infection already has occurred but is at a sufficiently earlystage that the immune response produced to the vaccine effectivelyinhibits further spread of infection.

In a preferred embodiment, the whole cell vaccine is administered to afemale animal to prevent or treat the occurrence of mastitis. In thisregard it is administered to female animals considered to be at risk forthe development of mastitis, i.e., to animals used in a breedingprogram, and more particularly to those animals with a prior history ofmastitis. The whole cell vaccine is particularly useful in preventingmastitis in a farm animal, such as a cow or a sow, although it also isindicated for use in pet animals, such as dogs and cats.

By another approach, a vaccine of the present invention can beadministered to a subject who then acts as a source for immuneglobulin,produced in response to challenge from the specific vaccine(“hyperimmune globulin”), that contains antibodies directed against S.aureus. A subject thus treated would donate plasma from whichhyperimmune globulin would then be obtained, via conventionalplasma-fractionation methodology, and administered to another subject inorder to impart resistance against or to treat S. aureus infection.Hyperimmune globulins according to the invention are particularly usefulfor immune-compromised individuals, for individuals undergoing invasiveprocedures or where time does not permit the individual to produce hisown antibodies in response to vaccination. Hyperimmune globulinsproduced to whole cell vaccine are particularly useful in treating S.aureus infections.

Similarly, monoclonal or polyclonal anti-S. aureus antibodies producedaccording to the present invention can be conjugated to an immunotoxin,and administered to a subject in whom S. aureus infection has alreadyoccurred but has not become widely spread. To this end, antibodymaterial produced pursuant to the present description would beadministered in a pharmaceutically acceptable carrier, as definedherein.

The present invention is further described by reference to thefollowing, illustrative examples.

EXAMPLES Example 1

Fermentation of S. aureus

A strain of S. aureus that carries the 336 antigen was cultivated inColumbia broth supplemented with 2% NaCl in an 80-liter fermentorcontaining 60 liters of broth medium at 370°. The fermentation wasstarted with one liter of a 16 hour old seed culture. The cells weregrown with agitation at 200 rpm for 24 hours, to an A_(650nm) of 20.0.

Cells to be used as a vaccine to prepare whole cell antiserum wereformalin fixed overnight at room temperature. Cells for purification ofantigen were killed by adding phenol-ethanol (1:1, vol/vol) to thefermentor to a final concentration of 2%, and mixing slowly for 2 hoursat 15-20° C. No viable cells were detected after this treatment. Thecells then were harvested by centrifugation at 14,500×g and stored at−70° C. until use. Approximately 800-900 grams of cell paste (netweight) was obtained from a 60-liter fermentation.

For preparation of cells to be used in preparation of a whole cellvaccine, strains of S. aureus that carry the 336 antigen, the Type 5antigen and the Type 8 antigen were separately cultivated in Columbiabroth supplemented with MgCl₂/CaCl₂ at 37° for 18-20 hours, whileshaking (200 rpm). The cells were tested by slide agglutination fornegative reactivity with S. aureus Wood strain antisera, and thenincubated with 3% formalin for 24 hours. The cells were washed 3 timeswith PBS, typed with S. aureus specific antibodies (T5, T8 or 336) andWood strain antisera and adjusted to appropriate OD at 540 nm.

Example 2

Preparation of Whole Cell Vaccine and Antiserum

Formalin-fixed cells from Example 1 were adjusted at OD_(540 nm)=1 andcombined with physiological saline to produce a vaccine. No adjuvant wasused. The vaccine was injected intravenously into rabbits. Rabbits werebled at weekly intervals and positive whole cell serum was collected andpooled. IgG was purified from whole cell serum by a protein G affinitycolumn. The purified material contained 23 mg/ml total IgG (280 UV scan)and substantially less 336 antigen-specific IgG.

Example 3

Purification of 336 Antigen

The cell paste was suspended at 0.5 g (wet weight) per ml in 0.05 MTris-2 mM MgSo₄, pH 7.5. Lysostaphin (100 to 150 μg/ml) was added andincubated at 37° C. for 3 hours with mixing. Thereafter, DNase and Rnasewere added to final concentrations of 50 μg/ml each, and the incubationwas continued for an additional 4 hours. The reaction mixture wasprecipitated sequentially with 25 and 75% ethanol in the presence of 10mM CaCl₂.

The 75% ethanol precipitate was pelleted by centrifugation at 12,000×gfor 30 minutes, or at a lower rpm for a longer time. The supernatant wastransferred to dialysis tubing. The reaction mixture was filteredthrough a 0.45 μm pore-size membrane and precipitated sequentially with25 and 75% ethanol in the presence of 10 mM CaCl₂. The 75% ethanolprecipitate was dialyzed extensively against water at 3 to 8° C. andfreeze-dried. The powder was dissolved in 0.2 M NaCl/0.05 M Tris HCl, pH7.0. The resulting crude material was loaded onto a Q Sepharose columnin 0.2 M NaCl/0.05 M Tris HCl, pH 7.0, and eluted with a 0.2-0.4 M NaCllinear gradient. Fractions that contained antigen, as detected bycapillary precipitation with antiserum from Example 2, were pooled,dialyzed, and freeze-dried. Most of the antigen eluted at 0.32-0.35 MNaCl/0.05 M Tris HCl.

The crude antigen thus obtained was treated with 1 mg lysozyme per 10 mgcrude antigen in 10 mM CaCl₂ to digest residual peptidoglycancontamination. The lysozyme-treated crude antigen then was furtherpurified on a Sephacryl S-300 gel filtration column in 0.2 M NaCl/PBS 1×to obtain substantially pure antigen. All reactive material was screenedusing whole antiserum.

Example 4

Characterization of Antigen

Analysis of purified 336 antigen by gas liquid chromatography (GLC)shows the presence of glucosamine as a major glycosyl component. This isconfirmed by sugar analysis on the Dionex system. 1H-NMR spectroscopy ofthe 336 antigen shows one anomeric proton at 4.751 ppm, corresponding toβ-linked hexosamine. In addition, the NMR spectrum shows well separatedsignals at 4.229 ppm (2H), 3.649 (1H), 3.571 ppm (2H), 2.19 ppm (3H).Signals corresponding to O-acetyl groups are not found. This indicatesthe absence of O-acetylation, and is clearly distinguished from the20-80% O-acetylation found on other S. aureus type isolates, such asType 5 and Type 8. The 13C-NMR spectrum shows one signal in the anomericregion at 102.396 ppm. This confirms the presence of monosaccharide as amajor component. Other C13-NMR spectrum signals appear at 81.865,76.641, 74.950, 71.841, 71.051, 70.775, 67.665, 67.142, 61.716, 56.552,50.355, 43.408 and 23.246 ppm, respectively.

The mobility of purified antigen in immunoelectrophoresis (IEF)indicates the presence of negatively-charged groups. The purifiedantigen does not contain neutral sugars as detected by the phenolsulfuric assay. The K_(d) of purified antigen was 0.3 on Superose 12 HRcolumn, which is a smaller molecular size material in comparison withType 5 (K_(d) of 0.017), Type 8 (K_(d) of 0.061) and teichoic acid(K_(d) of 0.18).

Example 5

Antigen-Immunocarrier Conjugates

Purified antigen was derivatized with 0.5 M adipic acid dihydrazide(ADH) using 100 mm 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC)at pH 5.6. Percentage derivatization was achieved in the range of 2 to7% (w/w). Derivatized purified antigen was conjugated torecombinantly-produced, non-toxic mutant strain of Pseudomonasaeruginosa exotoxin A using 50 mM EDAC at 1:1 (antigen:protein), asdescribed in Fattom et al., Inf. and Imm. 60:584-589 (1992). Theconjugation yield was 50-70%, determined by measurement of protein. TheKd of the conjugate was 0.2 on the Superose 12 HR column.

The conjugate was injected into rabbits with adjuvant (CFA followed byIFA) at a 1:1 ratio. Positive bleeds were combined and IgGs werepurified on a protein G column. Conjugate-raised IgG showed identitywith antibodies induced by whole cell IgG against the antigen in animmunodiffusion assay. Purified conjugate sera IgG was shown to contain12.2 mg/ml total IgG by a 280 nm UV scan and 0.7 mg/ml antigen-specificIgG by ELISA. Whole cell serum, whole cell IgG, and conjugate IgG wereused in opsonophagocytosis assays and animal models.

Example 6

In vitro Opsonophagocytosis Assays with Antibodies to Antigen/ConjugateVaccine

Polymorphonuclear leukocytes (PMNs) were obtained from HL-60 cellsadjusted to a concentration of 1.0×10⁷ cells per ml in MEM supplementedwith 10% fetal bovine serum (FBS). S. aureus was grown overnight inColumbia broth supplemented with MgCl₂/CaCl₂. The concentration ofbacteria was adjusted spectrophotometrically to an OD of 0.02 at 540 nm(4×10⁶ cells/ml) then adjusted to 1×10⁶ cells/ml in MEM supplementedwith 10% FBS. Purified antigen-specific or control non-reactive IgGswere added to facilitate opsonization by PMNs. Baby rabbit complement,diluted 1:8 in MEM supplemented with 10% FBS was used as the negativecontrol.

The reaction mixture contained 25 μl S. aureus (concentration 1×10⁶cells/ml), 25 μl PMNs (concentration 1×10⁷ cells/ml), 25 μl complement,100 μl sera or antibodies, and sufficient MEM/10% FBS to bring the totalreaction volume to 250 μl. At 0 hours, 1 hours and 2 hours, 25 μl ofsample were serially diluted. 25 μl of the 10⁻², 10⁻³, 10⁻⁴ and 10⁻⁵dilutions were plated onto TSA agar plates, and incubated overnight at37° C.

The results are shown in FIG. 2, and show that antibody to conjugatemediates opsonophagocytosis of a representative strain of S. aureus thatcarries the 336 antigen. The results are reported as percent killing byamounts of 336 antigen-specific IgG ranging from 300μg to 128 μg. Forcomparison, percent killing by an equivalent amount of non-reactive IgGis also reported. PMNs plus complement was used as a control.

Example 7

In vitro Opsonophagocytosis Assays with Antibodies to Whole Cell Vaccine

To evaluate the efficacy of S. aureus 336 whole cell vaccine, the S.aureus 336 whole cell IgGs were tested for their role to mediateopsonophagocytic killing of S. aureus 336 strains in a whole bloodopsonophagocytic assay. S. aureus 336 cells were grown and adjusted to2×10⁶ cfu/ml, as described in Example 1. Bacterial suspension (100 μl),saline (100 μl) and antibody solution (100 μl) were added to 700 μl ofcitrated human blood. After mixing, an aliquot of 25 μl was taken andplated to obtain bacterial counts at time zero. The mixture then wasincubated with shaking at 37° C. for one hour and plated. The resultsare plotted in FIG. 3, and showed a 2 log reduction of colony formingunits in the presence of immune IgGs in citrated whole blood, ascompared to nonimmune IgGs. Activity of immune IgGs was the same even ata 1:4 dilution, indicating that supplementation of rabbit whole bloodwith antibodies generated against S. aureus 336 isolate produced astrong opsonophagocytic effect against S. aureus 336 strains.

Example 8

In vitro Opsonophagocytic Killing Mediated by Whole Cell HyperimmuneAntisera

The importance of S. aureus 336 specific antibodies in S. aureus 336whole cell derived hyperimmune rabbit antisera to mediateopsonophagocytic killing of S. aureus 336 bacteria was tested byabsorption of 336 specific antibodies from S. aureus 336 whole cellhyperimmune rabbit antisera with 336 antigen and T5 CPS antigen.Nonimmune rabbit serum was used as a control.

Frozen beads of a S. aureus 336 strain (91-14) was inoculated to 5 mL ofColumbia Mg/CaCl₂ broth and incubated for 16 hours at 37° C. with 200rpm shaking. For the assay, cells were then adjusted with saline to anapproximate concentration of 2×10⁶ CFU/mL (OD₅₄₀=0.02). PMNs (HL-60cells) were used at concentration 1×10⁷ cells/mL in 1×MEM opsonizationmedia [Minimum Essential Medium, with Earle's salt, without glutamate,GIBCO-BRL], supplemented with 0.1% gelatin [Sigma Chemicals]. Rabbitserum diluted 1:100 with opsonization media was used as a complementsource.

Absorption of S. aureus 336 whole cell antisera (heat inactivated for 30minutes at 56° C.) with S. aureus 336 antigen and T5 CPS antigen wasdone as follows. The antigens were dissolved in dH₂O at concentrationsranging from 250 ug/mL to 15.6 ug/mL. Antigen solutions (250 uL) withdifferent concentrations of the antigens were mixed with 250 uL of S.aureus 336 whole cell antisera diluted to 1:250. The reaction mixturewas first incubated for 2 hours at 37° C. and than overnight at 40C. Thesamples were then centrifuged at 14,000 rpm on an Eppendorf 5415centrifuge at room temperature. Supernatants were then used in theopsonization assay.

To initiate the assay, 50μl of bacteria, 50μl of diluted complement, 5μlof adjusted HL-60 cells and 50μl of antisera were added per well inpolystyrene round bottom micro titer plates (Corning Glass Works).Following mixing, a 25 μl aliquot was taken for determination of Time 0bacterial counts. The plate was centrifuged for 5 minutes at 1200 rpm at37° C. and incubated for an hour in 5% CO₂ in an incubator. A 25 ulaliquot was taken for determination of CFU/mL of bacteria at one hour(T₁).

Bacterial suspensions were diluted 1:10, 1:100, 1:500, 1:1,000 and1:2,000 in distilled water, and 20 μl of the last four dilutions wereplated onto TSA agar plates. Emerging colonies from these plates wereused to quantify percent survival by following formulae:${\% \quad {kill}} = {\frac{{CFU}\text{/}{mL}\quad {at}\quad T_{1}}{{CFU}\text{/}{mL}\quad {at}\quad T_{0}} \times 100\%}$

The results are summarized in FIG. 4 and show that S. aureus 336hyperimmune sera reduced bacterial growth more then 90%, while nonimmunerabbit antisera did not mediate opsonophagocytic killing as compared tocontrol containing no antisera (complement, PMNs and bacteria, denotedC+P+B). Preincubation of antisera with different amounts of S. aureus336 antigen and T5 CPS antigen showed that opsonophagocytic killing ofS. aureus 336 whole cell sera is mediated mostly by 336PS specificantibodies. Absorption with T5 CPS antigen did not change theopsonophagocytic killing of 336 whole cell antisera. On the other hand,there is a reduction in opsonophagocytic killing following absorptionwith 336 antigen, with the degree of reduction correlating to amount of336 antigen used in the absorption. Inhibition was titered out at lowamounts of added 336 antigen. S. aureus 336 whole cell immunization didnot induce formation of T5 CPS specific or cross-reacting antibodies asshown by the fact that T5 CPS could not absorb the opsonophagocytickilling, even when 62.5 ug of T5 CPS was used for absorption.

What is claimed is:
 1. A vaccine that comprises (A) cells, cell lysates,or cell derivatives of Staphylococcus aureus which carry an antigen that(a) comprises β-linked hexosamine, (b) contains no O-acetyl groupsdetectable by nuclear magnetic resonance spectroscopy, and (c)specifically bind with antibodies to Staphylococcus aureus Type 336deposited under ATCC 55804, and a pharmaceutically acceptable carrier.2. An immunotherapy method comprising administering to a subject animmunostimulatory amount of composition as claimed in claim
 1. 3. Animmunotherapy method according to claim 2, wherein said subject is afemale animal at risk for developing mastitis.
 4. An immunotherapymethod according to claim 3, wherein said female animal is a cow.
 5. Animmunotherapy method according to claim 3, wherein said female animal isa sow.
 6. An immunotherapy method according to claim 3, wherein saidfemale animal is a cat.
 7. An immunotherapy method according to claim 3,wherein said female animal is a dog.
 8. An immunotherapy methodaccording to claim 2, wherein said subject is female animal withmastitis.
 9. An immunotherapy method according to claim 8, wherein saidfemale animal is a cow.
 10. An immunotherapy method according to claim8, wherein said female animal is a sow.
 11. An immunotherapy methodaccording to claim 8, wherein said female animal is a cat.
 12. Animmunotherapy method according to claim 8, wherein said female animal isa dog.
 13. A method of preparing an immunotherapeutic agent against S.aureus infection, comprising: immunizing subjects with a compositionaccording to claim 1; collecting plasma from said immunized subjects;and harvesting antibodies directed against S. aureus from said collectedplasma.
 14. An immunotherapeutic agent against S. aureus infection,comprising antibodies prepared by the method of claim 13 in apharmaceutically-acceptable carrier.
 15. A vaccine according to claim 1,additionally comprising cells, cell lysates, or cell derivatives ofStaphylococcus aureus which carry Type 5 antigen, and cells, celllysates, or cell derivatives of Staphylococcus aureus which carry Type 8antigen.
 16. A method of preparing an immunotherapeutic agent against S.aureus infection, comprising: immunizing subjects with a compositionaccording to claim 15; collecting plasma from said immunized subjects;and harvesting antibodies directed against S. aureus from said collectedplasma.
 17. A method of preparing a whole cell vaccine according toclaim 1, comprising: selecting cells of Staphylococcus aureus whichcarry an antigen that (a) comprises β-linked hexosamine, (b) contains noO-acetyl groups detectable by nuclear magnetic resonance spectroscopy,and (c) specifically bind with antibodies to Staphylococcus aureus Type336 deposited under ATCC 55804; inactivating said cells; and mixing saidcells, or lysates or derivatives of said cells, with pharmaceuticallyacceptable carrier.
 18. A vaccine, prepared the method of claim
 17. 19.A method of preparing a whole cell vaccine according to claim 17,wherein said cells are selected using antibodies to said antigen.
 20. Amethod of preparing a whole cell vaccine according to claim 19, whereinsaid antibodies are monoclonal antibodies prepared using isolatedantigen.
 21. A vaccine, prepared by the method of claim
 19. 22. Animmunotherapy method comprising administering to subject animmunostimulatory amount of a composition as claimed in claim
 19. 23. Animmunotherapy method according to claim 22, wherein said subject is afemale animal at risk for developing mastitis.
 24. An immunotherapymethod according to claim 22, wherein said subject is a female animalwith mastitis.
 25. A method of preparing an immunotherapeutic agentagainst S. aureus infection, comprising: immunizing subjects with acomposition according to claim 19; collecting plasma from said immunizedsubjects; and harvesting antibodies directed S. aureus from saidcollected plasma.
 26. An immunotherapeutic agent against S. aureusinfection comprising antibodies prepared by the method of claim 25 in apharmaceutically-acceptable carrier.